Rapid assay for detection of endotoxins

ABSTRACT

The presently claimed invention is an apparatus and method for the detection of endotoxin via a competitive assay.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of parent patent applicationentitled OPTICAL IMMUNOASSAY FOR MICROBIAL ANALYTES USING NON-SPECIFICDYES of Frances S. Ligler et al. designated by Ser. No. 08/102,933 andNavy Case No. 75,315 and filed in the U.S. Patent and Trademark Officeon Aug. 6, 1993now U.S. Pat. No. 5,496,700, which parent application isstill pending before the U.S. Patent and Trademark Office, and whichpatent application is incorporated herein by reference in its entiretyand for all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an apparatus and method for detectingendotoxin.

2. Description of the Related Art

All gram-negative bacteria and many fungi have endotoxin as a majorconstituent of their cell surface. Most gram-positive bacteria have asimilar, endotoxin-like molecule as a constituent of the cell surface.In general, bacterial endotoxins partially consist of a highly variableouter region and a conserved inner core. The variable outer region iscomposed of repeating oligosaccharide (sugar) units comprising theO-ANTIGENIC region. The OUTER CORE and the O-ANTIGEN region, foundwithin the cell walls of various gram negative bacterial species, showspecies differentiation among endotoxins. Bacterial endotoxins alsoconsist of a relatively constant, conserved inner core region. Theconserved inner core consists of the KDO region and the heptose region.The lipid A moiety (also a conserved portion of the endotoxin) is thetoxic region of the endotoxin found in the cell surface of gram-negativebacteria. An exemplary endotoxin is shown below: ##STR1##

Endotoxin is an extremely powerful stimulator of the immune system. Thedevastating effects of bacterial infections and septicemia are in largepart due to endotoxin. Mortality rates, due to septicemia are high, 60%or more. The most effective treatment of endotoxin related problems isearly detection in real time with high sensitivity. Additionally,endotoxin is a significant contaminant in food products andpharmaceutical products. Accurate determination of endotoxinconcentration is required prior to product distribution. To satisfy therequirements of industrial production, endotoxin assays must beaccurate, rapid and cost effective.

Levin and Bang observed in the horseshoe crab (Limulus polyphemus) thatblood coagulation was a consequence of gram-negative bacterialinfections. See Levin, J., Bang, F.B., 19 THROMBOS. DIATH. HAEMORRH.,186-197 (1968), incorporated by reference herein in its entirety and forall purposes. When an extract of the horseshoe crab blood was preparedand tested, a gelation reaction was observed in the presence ofendotoxin. Levin and Bang postulated that endotoxin mediated thegelation reaction of horseshoe crab blood. They further postulated thatthe gelation reaction was initiated enzymatically. The enzymeresponsible for initiating the gelation reaction was identified aslimulus amebocyte lysate (LAL).

The reaction mechanism for the gelation reaction in horseshoe crab bloodinvolves the activation of a proclotting enzyme by Ca⁺ and endotoxin.The activated proclotting enzyme catalyzes the hydrolytic cleavage ofcoagulogen (a clottable protein of 215 amino acid residues) intopolypeptide subunits. Clotting occurs following the cleavage of the 215amino acid coagulogen protein into a soluble peptide of 45 amino acidresidues and an insoluble peptide (coagulin) of approximately 170 aminoacid residues. The insoluble 170 amino acid peptide, coagulin, undergoespolymerization to form a stable clot or gel. The presence of a gel orclot indicates the presence of endotoxin. The formation of a gel or clotis used in what is known as the limulus amebocyte lysate method (LAL).The LAL method and its variations are the most commonly used endotoxindetection methods.

The standard LAL tests are of two types, gelation and chromogenic. Bothassays are based on the enzyme cleavage reaction of coagulogen, but, inthe chromogenic assay, a color is produced during the cleavage step.Thus, the presence of the color instead of a clot or gel indicates thepresence of endotoxin.

The limitations of these LAL assays include limitations of specificity,limitations of interfering substances and limitations ofreproducibility. The LAL techniques require an enzymatic reaction todetect the presence of endotoxin. Hence, substances that inhibit orstimulate enzymatic cleavage of the 215 amino acid coagulogen proteinwill lead to false-negative or false-positive results, respectively. Insamples, such as serum or blood, there are several factors known tointerfere with the LAL method. For example, the LAL enzyme cascade isinhibited by antibiotics, hormones, heavy metals, amino acids,alkaloids, carbohydrates, plasma proteins, enzymes, electrolytes andB-1,3-D-glucan. See Satoshi, M., Masahiro, N., Taizo, W., Tadashi, S.and Tetsuya, T., 198 ANALYTICAL BIOCHEMISTRY 292-297 (1983),incorporated by reference herein in its entirety and for all purposes.For example, false positive gelation can be caused by thrombin,thromboplastin, RNA, RNAase, trypsin, trypsin-like enzymes, lipotechoicacid and peptidoglycan fragments. False negative results (blockinggelation) can be caused by trypsin inhibitors, EDTA, other calciumbinding reagents, high salt concentrations and semi-syntheticpenicillins. See European Patent No. EP 0 265 127 A1, incorporatedherein by reference in its entirety and for all purposes.

Another detection strategy is the sandwich enzyme linked immunosorbentassay (ELISA). This assay (ELISA) involves immobilizing an antibodyspecific for a conserved region of the endotoxin (e.g. the KDO region).Using ELISA, the endotoxin immobilized (i.e. captured) by a firstantibody is detected by using a second antibody attached to anotherantigenic site of the endotoxin and a chromogenic enzyme. Thesensitivity of this technique is about 1 μg/ml. This test is timeconsuming, requiring over 4 hours.

There are several limitations of the ELISA assay used in the detectionof endotoxin. Endotoxin has low affinity for ELISA plates. The lack ofendotoxin affinity diminishes sensitivity. When a first antibody isimmobilized onto an ELISA plate and endotoxin is bound by (i.e. capturedby or immobilized by) the first antibody, there appears to besignificant interference with the binding of a secondary antibody to theimmobilized endotoxin, used in the detection of endotoxin. Finally, thesensitivity of ELISA assays (in the 1μg/ml range) is far below that ofthe LAL assays (1 ng/ml for chromogenic LAL assay) and higher thanclinically relevant concentrations of endotoxin of about 1 ng/ml.

Other variations of the LAL assays have been developed. These involvecombining the LAL assay with an enzyme linked immunosorbent assay(ELISA). A capture antibody (i.e. a polyclonal first anti-endotoxinantibody) for the oligosaccharide region of an endotoxin is immobilizedon a microtiter plate. Endotoxin is introduced over the ELISA microtiterplate. The bound endotoxin is detected by using the chromogenic LALsystem. Sensitivity is in the range of 2 pg/ml in PBS and 10 pg/ml indiluted plasma. See Mertsola, J., Cope, L.D., Munford R.S., McCracken,G.H. and Hansen, E.J, Detection of Experimnental Haemophilus influenzaeType b Bacteremia and Endotoxemia by Means of an Immunolimulus Assay,164 THE JOURNAL OF INFECTUOUS DISEASES 353-358 (1991), incorporated byreference herein in its entirety and for all purposes. See Mertsola etal., Specific Detection of Haemophilus influenzae Type bLipooligosaccharide by Immunoassay, 28 (12) JOURNAL OF CLINICALMICROBIOLOGY pp. 2700-2706 (December 1990), incorporated by referenceherein in its entirety and for all purposes. The combination ELISA/LALassay requires at least 12-24 hours to complete.

In the combination ELISA/LAL assay systems, the limitations arecumulative. The endotoxin is first bound to an ELISA microtiter platevia a reaction with an antibody or other capture molecule and theendotoxin is then detected using the chromogenic LAL assay. Thesubstances that interfere with the LAL assay also interfere with thecombination ELISA/LAL assay. In addition, the LAL reaction portion ofthe assay requires a minimum of 40 minutes to 2 hours to perform andrequires that serum be removed prior to the addition of the enzymebecause serum components may inhibit the enzyme activity. the LAL assaydoes not reliably quantitate the amount of endotoxin present. Detectionof endotoxin in serum is five times (5x) less sensitive than detectionof endotoxin in buffer.

An antibody-based test reported to have higher sensitivity than ELISA isthe latex immunoassay technique. In this assay, latex beads are coatedwith a monoclonal antibody (Ab1) specific for the O-9 determinant ofendotoxin. The beads are then incubated with a solution containinglipopolysaccharide (LPS; synonym for endotoxin). A magnetic bead coatedwith another monoclonal antibody (Ab2) specific for a differentantigenic site of an endotoxin is added to the LPS solution surroundingthe latex beads coated with Ab1. In the presence of LPS, the magneticbeads (coated with Ab2) complex with the latex beads (coated with Abl),via the LPS, and the latex beads are sedimented by the use of a magnet.The quantitation of LPS is based on the turbidity of the solutionremaining after sedimentation of the magnetic beads (i.e. measuring thelatex beads still remaining in solution after sedimentation). Thesensitivity varies based on incubation time from 5-30 minutes.Sensitivities of 0.9-25 ng/ml were reported. A serious disadvantage ofthis assay is that it is inhibited by serum and by high concentrationsof endotoxin. See Lim, P., 135 JOURNAL OF IMMUNOLOGICAL METHODS 257-261(1990), incorporated by reference herein in its entirety and for allpurposes.

U.S. Pat. No. 5,057,598 (Pollack et al.) discloses the use of monoclonalantibodies for the immunological detection of endotoxin or endotoxinbearing organisms. See Pollack et al. (U.S. Pat. No. 5,057,598),incorporated by reference herein in its entirety and for all purposes.Pollack et al. states, at column 18, lines 24-28, that detection ofendotoxin can be carried out in hours compared with detection ofendotoxin based on standard microbiological or cultural methods in days.Clearly, a detection method that works in a time shorter than hourswould be advantageous.

The oldest and best known test for endotoxin is the rabbit pyrogen test.This assay has a low sensitivity, is expensive and is plagued withreproducibility problems since different rabbits have differentsensitivities to endotoxin challenge. Additionally, animal tests arevery time consuming and, therefore, of limited application in a clinicalsetting.

The immunoassays for endotoxin previously described are all sandwichimmunoassays which include the binding of two proteins to the endotoxinmolecule. In general, sandwich assays are the preferred approach for thedetection of large molecule, whereas competition assays are used for thedetection of small molecules with only a single protein binding site.Another sandwich assay for endotoxin, reported by Connelly, useslipopolysaccharide binding proteins of amebocyte lysates and labelleddetection reagents. See U.S. Pat. No. 4,906,567, incorporated herein byreference in its entirety and for all purposes. The general scheme usedby Connelly involves holding lipopolysaccharide binding proteins fromone or more various organisms (See Col. 4, lines 62-68, U.S. Pat. No.4,986,567 of Connelly) within the wells of microtiter plates for about 2hours followed by washing in PBS (phosphate buffered saline), followedby holding BSA (bovine serum albumin) within the same microtiter platewells for about 1 hour, followed by introduction of an endotoxincontaining sample (or sample suspected of containing endotoxin) andholding the sample for about 30 minutes to about 1 hour within the samewells, followed by introduction of a horseradish peroxidase conjugatedto an LPS antibody via the heterobifunctional linking agentN-succinimidyl-4-(N-maleimidomehtyl)cyclohexane-1-carboxylate (SMCC) andholding the LPS antibody-peroxidase conjugate within the same wells forfor about 30-60 minutes, followed by washing in PBS and introducing achromogenic substrate, tetramethylbenzidine (TMB) into the same wellsand holding for about another 10-15 minutes before taking an opticaldensity measurment at 630 nm. See Connelly at Examples 1, 2, 3, 4 and 5.Id.

In all of the Connelly examples, the pH is maintained at 9.0 or less andthe time to reading the optical density (OD) from the time when thesample containing LPS (or suspected of containing LPS) is firstintrtoduced into the microtiter plate wells is between about 1 1/6 hours(1 hour, 10 min.--simultaneous or staggered addition of example 5 ofConnelly at Col 12, lines 15-35) to about 3 1/6 hours (3 hours, 10min.--sequential addition of example 4 of Connelly at Cols. 10 and 11).

European Patent (EP 0 265 127) of Harvey and Wilson describes a methodand apparatus for the detection of endotoxin using either polymyxin, anoctapeptin, or other similar cyclic peptides. An assay is carried outwherein the amount of a polymyxin-endotoxin conjugate (hereinafter,polymyxin B-LPS conjugate) formed is quantitated. The amount of thepolymyxin B-LPS conjugate formed is quantitated by attaching a label toeither the polymyxin B or to the endotoxin. The labelled polymyxin B-LPSconjugate is then measured. At page 7, lines 27-31 of EP 0 265 127 it isstated that:

In one form of the assay, the analyte which contains LPS and a standard,labelled, LPS preparation compete for a limited amount of immobilizedpolymyxin B, and the amount of label bound to the polymyxin B is thenquantitated. (Emphasis added.) From the above quoted language, itappears at first glance that the analyte (containing LPS or suspected ofcontaining LPS) and the standard, labelled, LPS preparation aresimultaneously placed in proximity to the immobilized polymyxin Bwherein the analyte LPS and the standard, labelled LPS compete forbinding to the polymyxin B. However, upon closer examination of Examples1, 2, 3, and 4 of EP 0 265 127, it appears that the analyte LPS and thestandard, labelled, LPS preparation are not added simultaneously.Instead, the analyte LPS and the standard, labelled, LPS preparation areadded consecutively in proximity to the exemplary immobilized polymyxinB. The requirement that the sample and labelled reagent be addedsequentially causes the assay to be inherently slower than an assayinvolving simultaneous addition of analyte and labelled endotoxin.Examples 1-4 describe slow, multistep reactions.

Example 1 of EP 0 265 127 describes a process wherein the followingsteps are executed:

(1) binding capacity of immobilized polymyxin B for LPS is determined byusing isotopically labelled LPS (¹⁴ C LPS);

(2) incubating test analyte solution (containing LPS or suspected ofcontaining LPS) with immobilized polymyxin B;

(3) adding a known quantity of isotopically labelled LPS (¹⁴ C LPS) tothe mixture of step (2);

(4) measuring the amount of isotopically labelled LPS in solution;

(5) subtracting the amount of isotopically labelled LPS in solution(i.e. unbound isotopically labelled LPS) from the total amount of ¹⁴ CLPS introduced in step (3) to determine the amound of ¹⁴ C LPS bound tothe immobilized polymyxin B; and

(6) subtracting the amount of ¹⁴ C LPS bound to the immobilizedpolymyxin B determined in step (5) from the binding capacity of theimmobilized polymyxin B in step (1) to determine the amount of analyteLPS present. The net result of step (6) indicates the amount of analyteLPS present and bound to the immobilized polymyxin B.

Example 2 of EP 0 265 127 describes a process wherein the followingsteps are executed:

(1) incubating analyte LPS samples with a known quantity of polymyxin Balkaline phospatase conjugate in sufficient excess to promote bindingbetween all the analyte LPS and the polymyxin B-alkaline phosphataseconjugate;

(2) incubating the mixture of step (2) with an immobilized, standard LPSpreparation to bind the unbound excess of the polymyxin B alkalinephosphatase conjugate from step (1);

(3) rinsing the preparation of step (2);

(4) measuring the amount of the excess polymyxin B-alkaline phosphataseconjugate of step (1) now bound to the immobilized, standard LPSpreparation of step (2); and

(5) subtracting the amount of excess polymyxin B alkaline phosphatasedetermined in step (4) from the total polymyxin B alkaline phosphataseconjugate used in step (1) to determine the amount of analyte LPSpresent. An assay time of 1 hour plus the time necessary forscintillation counting was required. Sensitivity was 10 μg/ml. Thestatement was made that increasing the specific activity of the ¹⁴ C-LPSwould increase sensitivity of the assay. However, increasing specificactivity also increases bacground so that the gain from such animprovement in the labelled reagent is rarely greater than a factor of10. In addition, radiolabels may be hazardous to an inexperienced userand involve undesirable problems of disposal as hazardous waste.

Example 3 of EP 0 265 127 describes a process wherein the followingsteps are executed:

(1) incubating a limited excess amount of immobilized polymyxin B withanalyte LPS to bind all analyte LPS;

(2) incubating an excess of standard, LPS-alkaline phosphatase conjugatewith the mixture of step (1) and removing excess standard LPs-alkalinephosphatase by rinsing;

(3) measuring the amount of standard LPS-alkaline phosphatase conjugatebound in step (2) to the immobilized polymyxin B; and

(4) subtracting the amount of the immobilized LPS-alkaline phosphataseconjugate bound in step (2) from the total amount of immobilizedpolymyxin B to determine the amount of analyte LPS bound in step (1). Itis difficult to imagine how Example 3 is characterized as a"displacement ELISA" (see p. 10, line 21) since it is stated at p. 8,line 25 that LPS can block the binding of ¹⁴ C LPS to polymyxin B/S4B.See EP 0 265 127.

Example 4 of EP 0 265 127 describes a process wherein the followingsteps are executed:

(1) immobilizing analyte LPS and rinsing;

(2) binding polymyxin B-alkaline phosphatase conjugate to immobilizedanalyte LPS of step (1); and

(3) measuring the amount of labelled polymyxin B attached to theimmobilized analyte LPS of step (1) to determine the amount of analyteLPS present in an analyte sample.

Examples 2-4 provide neither data nor experimental details. ELISA assaysare generally less sensitive than radioimmunoassays using the samereagents and same general approach. Thus, the approaches described inexamples 2-4 would not be expected to produce sensitivity greater thanabout 10 μg/ml. The use of enzymes as labels also involve problems ofinterferents from the sample matrix, increasing background response overtime during the assay, and increasing instability of the enzyme labelduring storage.

In all of the assay formats described, European Patent (EP 0 265 127)has several drawbacks:

(1) washing steps are required in Examples 2, 3, and 4;

(2) lengthy incubation steps are required in Examples 1, 2, 3, and 4, sothat all assays require more than 1 hour to perform;

(3) a radioactive label is used in example 1;

(4) enzyme labelled LPS or enzyme labelled polymyxin B is used inExamples 2, 3, and 4; and

(5) analyte samples used are extracts from, for example, body fluids(see p. 7, line 33 of EP 0 265 127). (6) no evidence of sensitivity orpotential sensitivity greater than μg/ml is provided.

Thus, there remains a need for an endotoxin assay with high sensitivityto concentrations as low as about 1 ng/ml which can be used innon-homogeneous samples such as serum or saliva, which has norequirement for enzymes or radiolabels, which requires very littlemanipulation by the operator, which is rapid, which can be used withintact cells, cell fragments or solubilized cells, which can be used ona wide variety of clinical and environmental samples, which requiresminimal or no sample preparation, which can be used in a wide variety ofenvironments, structural forms and conditions, which can be used rapidly(between about 15 seconds to about 10 minutes) to test for the presenceof endotoxin and which can be adapted to determine the specific type ofendotoxin detected.

SUMMARY OF THE INVENTION

It is, therefore, an object of this invention to provide a rapid testfor endotoxin.

It is another object of this invention to provide a rapid test forendotoxin which can be performed in the presence of serum or otherbiological samples including, but not limited to, urine, saliva, andmucus.

It is yet another object of this invention to provide a test forendotoxin which has no requirement for enzymes.

It is a further object of this invention to provide an assay requiringvery little manipulation by the operator.

It is yet a further object of this invention to provide a rapid test forendotoxin which is sensitive to quantities as low as 1 ng/ml.

It is even a further object of this invention to provide a rapid testfor endotoxin in intact cells, cell fragments or purified endotoxin.

It is an additional object of this invention to provide a rapid test forendotoxin in aqueous samples, for example, from the environment ormanufacturing processes.

It is an additional object of this invention to provide a rapid test forendotoxin in a wide variety of environments, structural forms andconditions.

It is an additional object of this invention to provide a rapid test forendotoxin and, if desired, to diagnose the specific type of endotoxindetected.

These and other objects of the presently claimed invention areaccomplished by a process which is fast, sensitive, requires no enzymelinked detection systems and can be performed successfully withnon-homogeneous samples containing endotoxin. The claimed assay relieson the binding of an endotoxin to a suitable capture molecule and thecompetitive detection of the capture molecule-endotoxin complex withoutthe use of enzymatic reactions to visualize or enhance detection. Forexample, a fluorescent dye may be used to label the capture-moleculeendotoxin complex and form an exemplary capturemolecule-endotoxin-fluorescent label complex. Detection of the exemplaryfluorescent label may be carried out by detecting a fluorescence signalobtained from the evanescent wave region of a fiber optic waveguideprobe wherein the capture molecule-endotoxin-label complex isimmobilized on the surface of the sensing portion of the fiber opticwaveguide probe.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 is a concentration-response curve obtained from the directbinding of increasing concentrations of fluorescently labelled E. coli0128:B12-endotoxin to a capture molecule, polymyxin B, the capturemolecule being covalently bound to the surface of a combination taperedfiber optic waveguide probe.

FIG. 2 is a concentration-response curve obtained from the binding ofincreasing concentrations of fluorescently labelled E. coli0128:B12-endotoxin to a capture molecule, goat IgG, the capture moleculebeing covalently bound to the surface of a combination tapered fiberoptic waveguide probe.

FIG. 3 is a concentration-response curve obtained from the binding of17ng/ml of fluorescently labelled E. coli EH100 Ra mutant endotoxin to acapture molecule, limulin lectin, the capture molecule being immobilizedon to the surface of a combination tapered fiber optic waveguide probe.The response is measured in microvolts (μVolts).

FIG. 4 is a concentration-response curve obtained from the binding ofincreasing concentrations of fluorescently labelled E. coli 0128:bl2endotoxin to a capture molecule, polymyxin B, the capture molecule beingimmobilized on to the surface of a combination tapered fiber opticwaveguide probe. The response is measured in microvolts (μVolts) whichresponse varies proportionately with the concentration of endotoxinpresent in the sample tested.

FIG. 5 is a concentration-response curve obtained from the competitivebinding of increasing concentrations of unlabelled E. coli 0128:bl2endotoxin in the presence of fluorescently labelled E. coli 0128:bl2endotoxin. The capture molecule was polymyxin B

FIG. 6 is a concentration response curve obtained from the binding ofincreasing concentrations of fluorescently labelled E. coli 0128:bl2endotoxin to a capture molecule, polymyxin B, the capture molecule beingimmobilized on to the surface of a fiber optic waveguide. The responseis measured in microvolts (μVolts) which response varies proportionatelywith the concentration of endotoxin present in the sample tested andeach point is an average of triplicate determinations.

FIG. 7 is a concentration-response curve obtained from the competitivebinding of increasing concentrations of unlabelled E. coli 0128:bl2endotoxin in the presence of fluorescently labelled E. coli 0128:bl2endotoxin. The capture molecule was polymyxin B.

DETAILED DESCRIPTON OF THE PREFERRED EMBODIMENTS

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. However,the following detailed description of the invention should not beconstrued to unduly limit the present invention. Variations andmodifications in the embodiments discussed may be made by those ofordinary skill in the art without departing from the scope of thepresent inventive discovery.

Broadly, the competetive assay of the presently claimed inventioncomprises:

(1) one or more endotoxin capture molecules immobilized upon asubstrate;

(2) a standard sample of one or more labelled endotoxin molecules;

(3) a means for detecting the presence of one or more endotoxin capturemolecule-labelled endotoxin molecule complexes (ECM-LEM complexes), saidone or more ECM-LEM complexes forming in the presence of one or moreunlabelled analyte endotoxin molecules (AEM); and

(4) a means for measuring the quantity of ECM-LEM complexes formed.

The capture molecules include but are not limited to antibodies,lectins, cell receptors, antibiotics, endotoxin binding proteins, orspecifically engineered peptides referenced in Random Peptide Libraries:A Source of Specific Protein Binding Molecules by J.J. Devlin et al.,published in SCIENCE, Vol. 24, pp. 404-405 (1990), incorporated hererinby reference in its entirety and for all purposes. Antibodies andantibiotics (such as polymyxin B) may be preferred simply because oftheir specificity, availability and stability following immobilization.

The capture molecule can be adsorbed or covalently bound to thesubstrate. Procedures for immobilizing capture molecules onto asubstrate (e.g. solid surface) are given in U.S. Pat. No. 5,077,210 ofLigler et al., incorporated herein by reference in its entirety and forall purposes.

Exemplary capture molecules, such as antibiotics, lectins, antibodies,and endotoxin binding proteins, are covalently immobilized on asubstrate carrying exemplary surface reactive groups such as hydroxylgroups. Exemplary substrates (e.g. solid supports) have or can bemodified to have surface reactive groups such as hydroxyl groups whichcan be reacted with a capture molecule for direct crosslinking or with asilane or alkyl thiol film for indirect crosslinking. The substrate ontowhich the capture molecule is immobilized depends only on the type ofsystem used to quantify the amount of unlabelled analyte endotoxinmolecules present. Exemplary substrates may include slides, beads(magnetic, synthetic or natural polymers), optical fibers, metal films,and cuvettes (quartz, glass, silica). The exemplary surface of thesubstrate may be smooth, flat, curved, round, rough, or with or withoutedges. Suitable substrates are preferably inorganic substrates includingbut not limited to silicon, glass, silica, quartz, metal oxides, organicpolymers, and the like which can be for example optical fibers, wires,wafers, films, discs or planar surface, microscope slides, or beads.Generally, the solid surfaces (substrates) have or can be modified tohave functional groups such as surface hydroxyl groups that react withexemplary silanizing reagents or exemplary metal oxide groups reactivewith exemplary alkyl thiol reagents.

Endotoxin has several chemically reactive groups including primaryamines and carboxyl groups. Such amine and carboxyl groups allow forlabel attachment at various sites on the endotoxin. In a preferredembodinent, the type of label used in the presently claimed inventionmust generate a signal at the surface of an optical waveguide. Examplesinclude fluorophores, colorimetric dyes, metal chelates or carbonyls,electrochemiluminescent labels, or luminescent labels. A label isselected so that a signal characteristic of a labelled endotoxinmolecule when the labelled endotoxin molecule is bound to an endotoxincapture molecule, forming an endotoxin capture molecule-labelledendotoxin molecule (ECM-LEM) complex, can be detected in both thepresence and absence of a competing endotoxin capturemolecule--unlabelled analyte endotoxin molecule (ECM-AEM) complex.

The immobilization of the ECM can be via covalent or noncovalentchemistries. The objective is to affix the ECM to a substrate surface insuch a way that the capture molecule retains its biological activitywhile remaining fixed to the substrate surface for the purpose ofquantitation. For example, immobilization of an exemplary endotoxincapture molecule (ECM) may be carried out upon the surface of theexemplary substrate having a surface coated with an exemplaryheterobifunctional crosslinking agent. For example, polymyxin B may beimmobilized onto the exemplary substrate (having a surface coated withan exemplary heterobifunctional crosslinking agent) at a concentrationof about 1-10 mg/ml of polymyxin B in an exemplary 0.1M NaBoratesolution at an exemplary pH of 9.0. The prepared exemplary substrate issuspended in the polymyxin B solution for about 30 min. The pH of theexemplary solution is then neutralized using HCI. The exemplarysubstrate is then washed several times with PBS to remove any unreactedexemplary polymyxin B and to further neutralize the exemplary substratesurface.

Once the exemplary substrate has an exemplary capture moleculeimmobilized upon its surface, the process of the presently claimedcompetitive assay is carried out. The process of the presently claimedinvention comprises:

(1) immobilizing one or more ECM upon a substrate;

(2) introducing at time =t₀, a standard sample of one or more labelledendotoxin molecules and an analyte sample containing or suspected ofcontaining one or more analyte endotoxin molecules over the one or moreimmobilized ECMs to form one or more ECM-LEM complexes and, if anyanalyte endotoxin molecules are present in the analyte sample, to formone or more endotoxin capture molecule--analyte endotoxin molecule(ECM-AEM) complexes;

(3) measuring at time =t₁ the amount of one or more ECM-LEM complexesformed wherein t₄ <(t₁ -t₀) <t₃ minutes wherein t₄ is a time =t₄ I t₃ isa time =t₃, wherein t₄ is about 0.05 to about 0.20 minutes and t₃ is atime selected from the group consisting of about 10 minutes, 5 minutes,4 minutes, 3 minutes, 2 minutes, 1 minute, 0.5 minute and 0.25 minute,respectively; and

(4) calculating the amount, if any, of the ECM-AEM complexes formed.

The formation of the ECM-AEM complexes at the surface of a waveguide ismeasured using the waveguide to collect the signal generated by thelabel. The signal can be a change in refractive index, a phase shift inthe light, fluorescence, luminescence, absorbance, respectively.Depending upon the type of signal generated, various means ofquantitating the signal known in the art may be used. The device may ormay not need a light source to excite the label. If needed, theexcitation light can travel down the waveguide or excite the light fromoutside the waveguide.

Having described the invention, the following examples are given toillustrate specific applications of the invention, including the bestmode now known to perform the invention. These specific examples are notintended to limit the scope of the invention described in thisapplication.

EXAMPLES Fluorimeter and Waveguide Configuration used for Examples

The fluorimeter and waveguides used in the examples described here werelaboratory prototypes. A similar fluorimeter operating at 635 nm iscommercially available (Research International, Woodinville, WA).Exemplary suitable dyes for use in conjunction with fluorimeter fromResearch International are the sulfoindocyanine dyes (cyanine based dyese.g. Cy5 dyes) described by Mujumdar et. al. in the paper entitledCyanine Dye Labeling Reagents: Sulfoindocyanine Succinimidyl Esters,BIOCONJUGATE CHEMISTRY, Vol. 4, No. 2, pp105-111 (March/April1993)--incorporated herein by reference in its entirety and for allpurposes, the dyes being available from Biological Detection Systems,Inc. located in Pittsburgh, Pennsylvania. For details of the device andwaveguide construction, see L.C. Shriver-Lake, G.P. Anderson, J.P.Golden and F.S. Ligler, The effect of Tapering the Optical Fiber onEvanescent Wave Measurements 25 ANALYTICAL LETTERS 7, pp. 1183-1199(1992), incorporated by reference herein in its entirety and for allpurposes. See J.P. Golden, L.C. Shriver-Lake, G.P. Anderson, R.B.Thompson and F.S. Ligler, Fluorometer and Tapered Fiber Optic Probe forSensing in the Evanescent Wave 31 OPTiCAL ENGINEERING No. 7, pp.1458-1462 (July 1992), incorporated by reference herein in its entiretyand for all purposes. See G.P. Anderson, J.P. Golden and F.S. Ligler, AFiber Optic Biosensor: Combination Tapered Fibers Designed for ImprovedSignal Acquisition, 8 BIOSENSORS & BIOELECTRONICS, pp. 249-256 (1993),incorporated by reference herein in its entirety and for all purposes.

The detection optics in the fluorimeter used in these experiments wereencased in a light-proof metal enclosure to reduce the effects ofambient light and electromagnetic influence on the detector circuitry.Key components include optics for launching and collecting the lightmounted on kinematic mounts. A laser light source was selected for itsmoderate power, stability, narrow excitation bandwidth and efficientlight coupling into the fiber. The exemplary rhodamine-based fluorescentlabels (e.g. TRITC) used with the sensor in the examples described herewere excited at 514 nm and emit in the 570 (+50 nm) nm range where thereis little intrinsic fluorescence in most clinical and environmentalsamples. A 514 nm laser beam from an air-cooled 50-mW argon ion laser(Omnichrome 532, Chino, California) was launched into the most proximalend of the cladded fiber. The laser was adjusted to a 12-mW output tominimize bleaching of the fluorophores bound to the distal end of theoptical fiber . The line filter (Melles Griot) removed plasma lines fromthe laser source. The laser beam passed through a chopper, a dichroicmirror and a spherical lens (f/i, one inch focal length bioconvex lens;Newport Corporation) onto the proximal end of the optical fiber.Approximately, 8 degrees of the fiber's numerical aperture of 23 degreeswere filled. The collected fluorescence signal from the distal end ofthe optical fiber traveled the reverse path to the dichroic mirror whereit was reflected through a longpass filter (KV550) onto a siliconphotodiode. The chopper and photodiode (EG&G Judson) were connected to alock-in amplifier (LIA, Stanford Research Systems, Sunnyvale, Calif.)and computer for phase sensitive detection via chopper controller. Thephotodiode was selected rather than a photomultiplier tube because oflow cost, reliability and compatibility with the lock-in amplifier. Thedata, measured as pV, were collected using the laptop computer.

The fiber optic waveguide used in the examples was made from a length ofstep-index plastic clad silica optical fiber (200 um diameter core,Quartz et Silice, Quartz Products, Tuckerton, Delaware) with a connectoron the proximal end to facilitate replacement and alignment. The distalend was modified to perform biochemical assays in the evanescent wave.At the distal end of the fiber, 12.5 cm of cladding was stripped awayfrom the core by removal of the buffer and cladding with a razor blade.Residual cladding was removed by immersing this end of the optical fiberin concentrated hydrofluoric acid (HF) for 1 minute. This distal endportion of the fiber was the sensing region on which the capturemolecules were immobilized. The combination taper probe (i.e. thecombination of a first short tapered section and a second longer,shallower tapered section) was prepared by slowly immersing the decladdistal end of the fiber into concentrated HF, using a computercontrolled stepper motor. Two to three centimeters of the distal, uncladend of the fiber was first lowered into the concentrated HF acid. Thedistal, unclad end of the fiber was further lowered at a constant rateof about 0.53 cm/minute for the next 9 cm to create the graduallytapered section . The distal, unclad fiber was even further lowered intothe concentrated HF acid for another 1 cm at a constant rate of about0.045 cm/minute to create the more steeply tapered section. Thisrelatively steep tapered section was tapered from the original 100 ,mradius down to 63 am. The taper dimensions were measured with acalibrated optical microscope. See G.P. Anderson, J.P. Golden and F.S.Ligler, A Fiber Optic Biosensor: Combination Tapered Fibers Designed forImproved Signal Acquisition, 8 BIOSENSORS & BIOELECTRONICS, pp. 250-251and FIG. 1 at pp. 250 (1993), incorporated by reference herein in itsentirety and for all purposes.

For immobilization of the capture molecules, the distal unclad taperedfiber having surface hydroxyl groups was immersed in a 2% solution ofmercaptopropyltrimethoxysilane (MTS) dissolved in toluene for 30 minutesunder N₂. Thereafter, the fiber was rinsed with toluene also under N₂.The silanized fiber was then 5 placed in a 2 mM solution of aheterobifunctional crosslinking agent, y-maleimidobutyryloxy succinimideester, for 1 h. The fiber was then rinsed in phosphate buffered saline(PBS) at pH 7.4. Lastly, the distal end of the fiber is suspended in asolution of 0.05 mg/ml of lectin, 0.05mg/ml solution of IgG antibody forendotoxin, 0.02 mg/ml of endotoxin neutralizing protein (ENP), 0.01,g/mlof polymyxin B, 0.1,ag/ml of polymyxin B, 1.0mg/ml of polymyxin B, or10.0mg/ml of polymyxin B, respectively, for 2 h. The fibers withimmobilized capture molecules are stored in endotoxin-free PBS at 4° C.See Bhatia et al., Analvtical Biochemistrv, 178, 408-413 (1989),incorporated herein by reference in its entirety and for all purposes.

After the immobilization of the capture molecules, the entire uncladdedportion of the exemplary combination tapered fiber optic waveguide wassealed into a flow chamber constructed from an exemplary shortened 200,l capillary tube and tee connectors, having a total length of 12 -13cm. The distal end of the fiber was glued outside the chamber, allowingonly light from the evanescent wave to enter the fiber surrounded by thesolution within the tee connection capillary. Both ends of the capillarytube were sealed with hot-melt glue, with the clad region of the fiberextending into the proximal portion of the apparatus. The sample to betested was introduced over the distal end of the optical fiber viasample inlet and exited at sample outlet. Use of this capillary tubeapparatus allows one to test a given analyte sample solution over theoptical waveguide and to wash the waveguide before introducing anotheranalyte sample solution.

Example 1

Labelled endotoxin was prepared and the binding of the labelledendotoxin to the capture molecule immobilized on the waveguide wasmeasured. In this example, polymyxin B was immobilized on the fiberusing a solution of 10mg/ml polymyxin B as described above.

A standard sample of labelled endotoxin molecules was made according tothe following procedure. A known amount of endotoxin (for example, 1mg/ml) was dissolved in 5.0 ml of a 0.1M NaBorate solution at pH 10.5and vortexed vigorously for at least 15 minutes at room temperature. Afluorescent label, tetramethyl isothiocyanate (TRITC, Sigma), was addedto the vortexed solution to a w/w concentration of 1:100 ofendotoxin:TRITC. The TRITC/endotoxin sample was incubated at 37 ° C. inthe dark for 4 h with intermittent vortexing . The TRITC labelledendotoxin was dialyzed against several changes of 0.15 M NaCl. Anyunbound TRITC was removed by passing the endotoxin solution over aSephadex G-25 column (Sigma). The standard sample of labelled endotoxinwas collected in fractions and the ratio of endotoxin to TRITCcalculated. Molar ratios of fluorophore to endotoxin of approximately0.8:1 to about 1.5:1 (i.e. molar conc. of fluorophore : molar conc. ofendotoxin) were considered acceptable for use in this example. Therewere two different endotoxins used, the E.Coli EH 100 Ra mutantendotoxin (lacking the 0-antigenic region) and from E.Coli 0128:B12endotoxin, respectively.

The TRITC labelled standard sample of endotoxin was further usedaccording to the following procedure. Labelled endotoxin wasreconstituted in PBS which contained either 2 mg/ml of bovine serumalbumin (BSA) or 0.1 % Triton X-114. The labelled endotoxin standardsample was introduced through the capillary tube containing the fiberoptic probe coated with the immobilized capture molecule. Binding wasobserved from about 0-2 minutes at 30 second intervals. The excitationlaser beam (514nm) was blocked between evanescent wave signalmeasurements to avoid photobleaching.

FIG. 1 depicts response curves obtained from the direct binding ofincreasing concentrations of fluorescently labelled E. coli 0128:B12endotoxin to a capture molecule, polymyxin B, the capture molecule beingcovalently bound to the surface of a fiber optic waveguide. Note thatthe fluorescent label used is tetramethyl rhodamine-5-isothiocyanate(TRITC). The labelled endotoxin standard was introduced through thecapillary tube containing the fiber optic waveguide coated with theimmobilized capture molecule. Binding signals were measured at 30 secondintervals. The excitation laser beam (514 nm) was blocked between signalmeasurements to avoid photobleaching. The signal measured from thestandard solutions of labelled endotoxin was determined using adifferent fiber waveguide for each recording. The response is measuredin microvolts (μVolts) which response varies proportionately with theconcentration of endotoxin present in the sample tested. The curves plotthe change in fluorescence at the waveguide surface as a function of theconcentration of fluorescently labelled E. coli 0128:B12-endotoxin insolution.

Example 2

In order (a) to demonstrate the binding of labelled endotoxin by adifferent capture molecule and (b) to demonstrate the quantitation ofsignal produced by increasing concentrations of the labelled endotoxin,waveguides coated with anti-endotoxin antibody were exposed toincreasing concentrations of labelled endotoxin.

FIG. 2 is a concentration-response curve obtained from the binding ofincreasing concentrations of fluorescently labelled E. coli0128:B12-endotoxin to a capture molecule, goat IgG antibody specific forE. coli 0128:B12 endotoxin, the capture molecule being covalently boundto the surface of a fiber optic waveguide. Note that the fluorescentlabel used is tetramethyl rhodamine-5-isothiocyanate (TRITC). Theresponse is measured in microvolts (μVolts) which response variesproportionately with the concentration of endotoxin present in thesample tested. The curve is a plot of change in fluorescence as afunction of the concentration of fluorescently labelled E. coli 0128:B12endotoxin in PBS +2 mg/ml BSA. The complex of antibody-endotoxin-TRITCwas detected as it formed at the waveguide surface.

Example 3

In order to demonstrate the binding of labelled endotoxin to yet anothercapture molecule on the surface of the waveguide, the waveguide wascoated with limulin lection. FIG. 3 is a time-response curve obtainedfrom the binding of 17 ng/ml of fluorescently labelled E. coli EhlOO Ramutant endotoxin to a capture molecule, limulin lectin, the capturemolecule being immobilized on to the surface of a fiber optic waveguide.The response is measured in microvolts (μVolts). The complex of limulinlectin-endotoxin-TRITC was detected as it formed at the surface of thewaveguide and within 1 minute of after the addition of the TRITClabelled endotoxin.

Example 4

In order to demonstrate a quantitative dose-response relationshipbetween the concentration of labelled endotoxin put over the waveguideand the signal produced, waveguides were coated with polymxyin B as thecapture molecule. FIG. 4 is a concentration-response curve obtained fromthe binding of increasing concentrations of fluorescently labelled E.coli 0128:B12 endotoxin to a capture molecule, polymyxin B, the capturemolecule being immobilized on to the surface of a combination taperedfiber optic waveguide probe. The response is measured in microvolts(μVolts) which response varies proportionately with the concentration ofendotoxin present in the sample tested. Under the conditions of theassay used to obtain FIG. 4, the capture molecule, polymyxin B, wasimmobilized onto the surface of a fiber optic waveguide probe and theassay was conducted in PBS +2 mg/ml BSA. The complex of polymyxinB-endotoxin-TRITC was detected as it formed at the surface of thewaveguide. The response shows percent signal (compared to the signalproduced on each fiber for 500 ng/ml of TRITC-endotoxin) as a functionof increasing concentrations of fluorescently labelled endotoxin from12.5 ng/ml to 500 ng/ml.

Example 5

Once the signal measured from the standard solution of labelledendotoxin was determined, the change in signal caused by a competitivebinding of unlabelled endotoxin and the standard endotoxin solution wasmeasured. Note that the subsequent signal was produced by a comingledsolution of the standard sample of labelled endotoxin and the analytesample of the unlabelled endotoxin flowing concomittantly over the fiberoptic probe held within the capillary tube, the probe being preparedusing polymxyxin B at 10 ng/ml.

FIG. 5 is a concentration-response curve obtained from the competitivebinding of increasing concentrations of unlabelled E. coli 0128:B12endotoxin in the presence of fluorescently labelled E. coli 0128:B12endotoxin. The fluorescently labelled and unlabelled endotoxin weredissolved in PBS +2 mg/ml BSA. The fluorescent label used was TRITC.Increasing concentrations of unlabelled endotoxin (0-250 ng/ml) wereadded to a standard concentration of TRITC-endotoxin of 200 ng/ml. Theresponse is measured in microvolts (μVolts) and each point is an averageof triplicate determinations and each point is standardized to %inhibition of signal from a 100% response (100% response is wherein nounlabelled endotoxin is present in solution and 200 ng/ml ofTRITC-endotoxin is present in solution of PBS +2 mg/ml BSA). Under theconditions of the competitive assay used to obtain FIG. 5, the capturemolecule, polymyxin B, was immobilized onto the surface of a fiber opticwaveguide probe and the assay was conducted in PBS +2 mg/ml BSA. Thecomplex of polymyxin B-endotoxin-TRITC was detected at the surface ofthe fiber optic waveguide. The response is standardized to show %inhibition of signal as a function of increasing concentrations ofunlabelled endotoxin from 0 to 250 ng/ml. Note that 10% of the expectedfluorescent signal (i.e. 100% response, supra) is inhibited at aconcentration of 12.5 ng/ml of unlabelled endotoxin in solution.

Example 6

The affect of serum on endotoxin binding and on the competitive assayfor endotoxin was determined according to the following procedure.

A concentration response curve obtained from the binding of increasingconcentrations of fluorescently labelled E. coli 0128:B12 endotoxin to acapture molecule, polymyxin B, the capture molecule being immobilized onto the surface of a combination tapered fiber optic waveguide probe isdepicted in FIG. 6. The response is measured in microvolts (μVolts)which response varies proportionately with the concentration ofendotoxin present in the sample tested and each point is the mean ±standard error of triplicate determinations. Under the conditions of theassay used to obtain FIG. 6, the capture molecule, polymyxin B, wasimmobilized onto the surface of a fiber optic waveguide probe and theassay was conducted in PBS +2 mg/ml BSA containing 0% serum, 1% serum,5% serum, or 20% serum. The complex of polymyxin B-endotoxin-TRITC wasdetected at the surface of the fiber optic waveguide. The figure showsthe percent signal as a function of increasing concentrations offluorescently labelled endotoxin from 12.5 ng/ml to 500 ng/ml. Note that25 ng/ml of endotoxin-TRITC can be detected in 20% serum and 12.5 ng/mlwas detected in 1% serum.

Example 7

FIG. 7 is a concentration-response curve obtained from the competitivebinding of increasing concentrations of unlabelled E. coli 0128:B12endotoxin in the presence of fluorescently labelled E. coli 0128:B12endotoxin. The fluorescently labelled and unlabelled endotoxin weredissolved in PBS +2 mg/ml BSA containing 0% serum, 1% serum, 5% serum or20% serum, respectively. The fluorescent label used was TRITC.Increasing concentrations of unlabelled endotoxin (0-250ng/ml) wereadded to a standard concentration of TRITC-endotoxin at a concentrationof 200 ng/ml. The response is measured in microvolts (μVolts) and eachpoint is an average of triplicate determinations and each point isstandardized to percent inhibition of signal from a 100% response (100%response is wherein no unlabelled endotoxin is present in solution and200 ng/ml of TRITC-endotoxin is present in solution of PBS +2 mg/mlBSA). Under the conditions of the competitive assay used to obtain FIG.7, the capture molecule, polymyxin B, was immobilized onto the surfaceof a fiber optic waveguide and the assay was conducted in PBS +2 mg/mlBSA. The complex of polymyxin B-endotoxin-TRITC was detected as itformed at the waveguide surface. The response is standardized to showpercent inhibition of signal as a function of increasing concentrationsof unlabelled endotoxin from 0 to 250 ng/ml. The curve for 0% serum (notshown) was identical to that for 1% serum. Note the expected fluorescentsignal (i.e. as compared to 100% response, supra) is inhibited at aconcentration of 25 ng/ml of unlabelled endotoxin in solution.

What is claimed is:
 1. A process for detecting endotoxin in aconcentration by a competitive assay, said process comprising the stepsof:(1) immobilizing polymyxin B upon a substrate under aphosphate-buffered saline solutions; (2) introducing at time =t₀, astandard sample of one or more labelled endotoxin molecules labelledwith TRITC (LEM) and an analyte sample containing or suspected ofcontaining one or more analyte endotoxin molecules over said one or moreimmobilized polymyxin B to form one or more polymyxin B--LEM complexesand, if any analyte endotoxin molecules are present in the analytesample, to form one or more polymyxin B--analyte endotoxin moleculecomplexes in phosphate-buffered saline; (3) measuring at time =t₁ theamount of one or more polymyxin B--LEM complexes formed wherein t₄ ≦ (t₁-T₀) ≦ t₃ minutes, wherein t₃ is a time =t₃ and wherein t₄ is a time =t₄; and (4) calculating the amount, if any, of the polymyxn B--analyteendotoxin molecule complexes formed.
 2. The method of claim 1, whereinsaid substrate is an optical fiber.